Welcome to the Molecular Machines A thread to lump together all the interesting discoveries regarding the intracellular biomolecular machinery that are crucial for life to exist. Please post interesting discoveries and perhaps describe the functionality of these intracellular biomolecular machines. Intracellular biomolecular machinery include the following: 1) DNA replication and repair machinery (replisome) 2) DNA transcription machinery and RNA processing and translation machinery (Spliceosomes and ribosomes) 3) Cell cycle signaling network (pRB, e2F, CDKs) 4) Programmed cell death machinery (Apoptosis, autophagy, mitotic catastrophe) 5) Protein processing machinery (Chaperones, ubiquitin-proteasome system) 6) Intracellular signaling networks (protein kinases and phosphatases) 7) Mechanical machines for intracellular shuttling of biomolecules and cellular movement (Microtubule network, kinesin, dynein) 8) Energy production machines (Electron transport chain, F0F1 ATP synthase) The Replisome: Sliding clamps, clamp-loaders and helicases. Sliding clamps are ring-shaped proteins that some refer to as the “guardians” of the genome or others name them as the “ringmasters” of the genome. Interestingly these clamps are structurally and functionally conserved in all branches of life and crystallographic studies have shown that they have almost superimposable three-dimensional structures, yet these components have very little sequence similarity (Figure 1) . Please Register or Log in to view the hidden image! Figure 1: Sliding clamps from the various domains of life. The picture below is taken from the Molecular Biology Visualization of DNA video (2:14) from the freesciencelectures.com site. Great video! Please Register or Log in to view the hidden image! Figure 2: Replication machinery. The following components can be seen: Sliding clamps (PCNA in eukaryotes): Green circular shaped Clamp loader (RFC in eukaryotes): Blue-white component in the middle (Figure 3: Structures of PCNA connected to RFC (front)) Please Register or Log in to view the hidden image! (Figure 4: Structures of PCNA connected to RFC (side)) (Figure 5: Structures of PCNA connected to RFC (back)) Helicase: Blue (Figure 6: Helicase (front)) DNA polymerase: Dark-blue components attached to the sliding clamps Primase: Green component attached to helicase Leading strand: Spinning off to the right Lagging strand: Spinning off to the top They are not ringmasters for nothing. Sliding clamps participate and control events that orchestrate DNA replication events in the following ways: Enhancement of DNA polymerase activity. Coordinate Okazaki fragment processing. Prevention of rereplication Translesion synthesis Prevents sister-chromatid recombination and also coordinates sister-chromatid cohesion Crucial role in mismatch repair, base excision repair, nucleotide excision repair Participates in chromatin assembly Other functions include: Epigenetic inheritance Chromatin remodeling Controls cell cycle and cell death signaling The true ringmasters. Clamp loaders are another group of interesting proteins (see video and figures 3-5 above). Interestingly again, their functional and structural architecture are conserved across the three domains of life with low-level sequence similarity . At the replication fork during replication, they load the sliding clamps many times onto the lagging strand (after DNA priming) and only once onto the leading strand. They also act as a bridge to connect the leading and lagging strand polymerases and the helicase. Which brings us to another interesting group of proteins; the helicases. Helicases are also known to be ring-shaped motor proteins, typically hexamers (see figure 6) and separate double-stranded DNA into single-stranded templates for the replication machinery. Replication occurs at about 1000 base pairs per second due to the highly efficient combination of sliding clamps and the polymerases. Thus, helicases need to unwind DNA at at least that speed. Unwinding DNA too slowly and the replication machinery might break down . Unwind the DNA too fast or untimely and harmful mutations might occur as single-stranded DNA is prone to degradation and cytosine deamination. The speed at which helicase unwinds DNA is no accident though, as it is intrinsically controlled. As helicase is bound to the lagging strand, it unwinds the leading strand in a separate direction. Applying a pulling force on the leading strand leads to a 7-fold increase in the speed of DNA unwinding by helicase [3, 4]. The highly efficient DNA polymerase/sliding clamp combination provides this controlling force on the leading strand. This forms a robust unwinding/polymerization interaction whereby polymerization controls and prevents unwanted DNA unwinding. Altogether, the replisome machinery provide a robust way for DNA replication to prevent unnecessary DNA damage and mutation. References. 1. Vivona JB, Kelman Z. The diverse spectrum of sliding clamp interacting proteins. FEBS Lett. 2003 Jul 10;546(2-3):167-72. 2. Jeruzalmi D, O'Donnell M, Kuriyan J. Clamp loaders and sliding clamps. Curr Opin Struct Biol. 2002 Apr;12(2):217-24. 3. Ha T. Need for speed: mechanical regulation of a replicative helicase. Cell. 2007 Jun 29;129(7):1249-50. 4. Johnson DS, Bai L, Smith BY, Patel SS, Wang MD. Single-molecule studies reveal dynamics of DNA unwinding by the ring-shaped T7 helicase. Cell. 2007 Jun 29;129(7):1299-309.